Search and Identification of Amyloid Proteins

Amyloids are fibrillar proteins with a cross-β structure. Pathological amyloids are associated with the development of a number of incurable diseases, while functional amyloids regulate vital processes. The detection of unknown amyloids in living objects is a difficult task, and therefore the question of the prevalence and biological significance of amyloids remains open. We present a description of two methods, the combination of which makes it possible to find and identify amyloid proteins in the proteome of various organisms. The method of proteomic screening for amyloids allows the detection of the proteins that form SDS-resistant aggregates. SDS resistance is a general feature of amyloid fibrils. Protein aggregates resistant to SDS treatment can be collected by ultracentrifugation and further identified by mass spectrometry. However, in addition to amyloids, SDS-resistant aggregates contain some non-amyloid proteins. To test the amyloid properties of proteins identified by proteomic screening, we developed the method of fibril immunoprecipitation followed by Congo red staining and birefringence analysis. The methods of proteomic screening and immunoprecipitation of fibrillar proteins have been successfully tested and applied for the identification of amyloid proteins in yeast and vertebrates.


Introduction
Amyloids are fibrillar proteins with a cross-beta structure. The formation of cytotoxic amyloid fibrils is often associated with the development of incurable systemic and localized amyloidosis [1]. At the same time, the so-called functional amyloids are characterized. This group includes proteins that are normally stored or function in the form of amyloid fibrils [2,3]. The search for and identification of proteins that form amyloid fibrils in various living objects is a rather difficult task. Most of the pathological and functional amyloids that have been characterized to date have been identified in the studies of a particular protein or a specific pathology. This approach does not allow assessment of the prevalence and biological significance of the pathological and functional amyloids in nature.
We have developed and successfully tested a PSIA-LC-MALDI method that detects proteins with amyloid properties in the proteomes of various organisms. The proposed method is based on the general feature of all known amyloid fibrils-their resistance to treatment with detergents such as SDS [4,5]. In contrast to amyloids, most non-amyloid protein aggregates and complexes are disassembled to monomers upon treatment with 1% SDS at room temperature. High molecular weight amyloid fibrils after SDS treatment can be separated from other proteins by ultracentrifugation. The proteins contained in this fraction are identified by mass spectrometry. The PSIA-LC-MALDI has been successfully used to identify proteins that form amyloid-like aggregates in yeast, bacteria, plants and vertebrate brain neurons [5][6][7][8][9][10]. It should be noted that some non-amyloid protein aggregates and complexes are also resistant to SDS treatment. Considering this, it is necessary to check whether the proteins identified in proteomic screening really form amyloid fibrils in vivo. Various techniques are used to test the amyloid properties of fibrillate proteins. In particular, the presence of a cross-beta structure, which is characteristic of amyloids, can be assessed using solid-state NMR [11], X-ray diffraction [12] or cryo-electron microscopy [13]. Unfortunately, in most cases, these methods apply only to the analysis of the purified protein in vitro. The customary way of evaluating protein amyloid properties in living organisms is to stain cytological preparations with amyloid-specific dyes, such as thioflavin T, thioflavin S or Congo red, and search for the colocalization of these dyes with the protein-specific antibodies [8,14,15]. In rare cases, these dyes can bind not just amyloid proteins [16][17][18]. Nowadays, staining of cytological preparations with Congo red, followed by the analysis of birefringence during polarization, is considered the most convincing proof of the amyloid nature of a protein [15]. Amyloids stained with Congo red exhibit a yellow-green birefringence in polarized light. At the same time, colocalization of the studied protein with an amyloid-specific dye cannot be final proof of its amyloid nature. Many proteins can be localized in the same area of a cytological preparation, and at such a level of resolution, it is impossible to conclude which of them binds Congo red. We have developed a method for the isolation of native amyloid fibrils from living organisms using immunoprecipitation for further evaluation of their amyloid properties ex vivo [8,19]. This method includes immunoprecipitation of the fibrillar protein detected in proteomic screening followed by the release of the protein from magnetic beads without destroying the fibrils. Fibrils are detected using electron microscopy and also stained with Congo red to assess birefringence.
The combination of methods of proteomic screening and analysis of amyloid properties of fibrillar proteins ex vivo enables the discovery of infectious amyloids (prions) and non-infectious amyloids in any organism. Using these approaches we identified functional amyloid FXR1 in mammalian brain neurons [5] and obtained direct evidence of the amyloid properties of yeast prions [19]. The development of these methods opens prospects for a systematic search for uncharacterized functional and pathological amyloids in various organisms.

Proteomic Screening for Amyloid Proteins
The method of proteomic screening of proteins that form amyloid-like aggregates (PSIA-LC-MALDI) includes several steps ( Figure 1). First, the protein lysate isolated from the tissues or unicellular organism is treated with 1% SDS at room temperature. This treatment facilitates the disassembly of most aggregates and complexes, while amyloid protofibrils remain intact and are not disassembled into monomers. At the next step, high molecular weight protein aggregates resistant to SDS treatment are separated from other proteins by ultracentrifugation. After washing and repeated ultracentrifugation, SDS is removed, and the samples are treated with trypsin. Peptides formed as a result of trypsinolysis are separated by HPLC and then identified by time-of-flight mass spectrometry. Based on these results, the mass spectrometer compiles a list of proteins present in the studied fraction.
The disadvantage of this method is that, in addition to amyloid proteins, other proteins that form SDS-resistant aggregates are also identified [6]. This disadvantage can be overcome if the analysis is carried out parallel to "a negative control". For example, a comparative study of proteins detected in proteomic screening in patients with suspected amyloidosis and patients of the control group can reveal proteins that aggregate only in pathology. For a search of functional amyloids in different organisms, the concentration of comparative study of proteins detected in proteomic screening in patients with suspected amyloidosis and patients of the control group can reveal proteins that aggregate only in pathology. For a search of functional amyloids in different organisms, the concentration of the detergent can be modified. For example, 2% SDS can be used instead of 1% for a more stringent selection of candidate proteins.

Immunoprecipitation of Amyloids and Ex Vivo Fibril Staining
To analyze the amyloid properties of proteins identified by proteomic screening, we developed an approach for isolating native fibrils from living organisms. At the first step, primary antibodies specific to the protein of interest are immobilized onto magnetic beads covered with protein A or G ( Figure 2). The binding specificity of A or G proteins to various primary antibodies has to be determined according to the manufacturer's protocol. At the next step, the protein lysate is incubated with protein A-or G-coupled magnetic beads. The magnetic particles are washed to get rid of unbound antibody material. In order to analyze the amyloid properties of the protein of interest, it is necessary to gently separate it from antibodies and magnetic particles, avoiding boiling. The resulting proteins are precipitated by ultracentrifugation and resuspended in water. Then, the fibrillar structure of the studied proteins can be analyzed using electron microscopy. Additionally, protein samples isolated by immunoprecipitation are stained with Congo red to analyze birefringence. This approach allows identification of both functional and pathological amyloids.
The disadvantage of this method is that in addition to the target protein, other proteins bound to amyloid fibrils are precipitated. That circumstance and the low amount of the precipitated protein complicate the further analysis of fibrils by physical methods that are used in the study of amyloids in vitro. Theoretically, after immunoprecipitation,

Immunoprecipitation of Amyloids and Ex Vivo Fibril Staining
To analyze the amyloid properties of proteins identified by proteomic screening, we developed an approach for isolating native fibrils from living organisms. At the first step, primary antibodies specific to the protein of interest are immobilized onto magnetic beads covered with protein A or G ( Figure 2). The binding specificity of A or G proteins to various primary antibodies has to be determined according to the manufacturer's protocol. At the next step, the protein lysate is incubated with protein A-or G-coupled magnetic beads. The magnetic particles are washed to get rid of unbound antibody material. In order to analyze the amyloid properties of the protein of interest, it is necessary to gently separate it from antibodies and magnetic particles, avoiding boiling. The resulting proteins are precipitated by ultracentrifugation and resuspended in water. Then, the fibrillar structure of the studied proteins can be analyzed using electron microscopy. Additionally, protein samples isolated by immunoprecipitation are stained with Congo red to analyze birefringence. This approach allows identification of both functional and pathological amyloids.
The disadvantage of this method is that in addition to the target protein, other proteins bound to amyloid fibrils are precipitated. That circumstance and the low amount of the precipitated protein complicate the further analysis of fibrils by physical methods that are used in the study of amyloids in vitro. Theoretically, after immunoprecipitation, the fibrils can be incubated with 1% SDS and precipitated by ultracentrifugation to eliminate other protein contaminants. In this case, it is necessary to isolate a large amount of the target protein so as not to lose it during additional treatments. the fibrils can be incubated with 1% SDS and precipitated by ultracentrifugation to eliminate other protein contaminants. In this case, it is necessary to isolate a large amount of the target protein so as not to lose it during additional treatments.     Add an equal volume of glass beads for disruption. 3.

Materials
Run 10 cycles of destruction of 20 s using benchtop homogenizer. In between cycles of destruction, incubate the samples for 1 min on ice.

4.
Transfer the cell lysate into new tubes and centrifuge at 805× g, 4 • C for 5 min.

5.
Transfer the supernatant (clarified cell lysate) into new tubes.  CRITICAL STEP Do not freeze lysates! Always work only with freshly prepared lysates.
Lysates from Animal Tissue 1. Homogenize tissue samples using a Cryogenic laboratory mill at −196 °C in liquid nitrogen according to Table 1. Lysates from Animal Tissue 1. Homogenize tissue samples using a Cryogenic laboratory mill at −196 °C in liquid nitrogen according to Table 1.

3.
Suspend 50 mg of homogenized tissue on ice in 0.5 mL of cold Lysis buffer 2 in 1.5 mL microcentrifuge tube. 4.

5.
Transfer the supernatant into new tubes.

6.
Use commercially available or made to order antibodies or serum that must meet the following requirements: • must recognize the native protein; • be suitable for immunoprecipitation.
The concentration of antibodies for immunoprecipitation is selected experimentally.

7.
Choose magnetic beads with immobilized protein A or G particles based on the type of antibodies you have (according to the manufacturer's recommendations). 8.
Wash the magnetic beads: • add 50 µL of suspension of selected magnetic beads to 200 µL of Binding buffer into a 2.0 mL microcentrifuge tube, mix gently; • using magnetic rack, let the particles settle on the magnet and carefully remove the supernatant with a pipette; • rewash the magnetic beads with 500 µL of Binding buffer using magnetic rack. 9.
Add 800 µL of Binding buffer, 8 µL of a Complete Protease Inhibitor Cocktail (×100) and preselected volume of antibody. 10. Incubate the mixture at room temperature for 1 h with slow overhead rotation.  Lysates from Animal Tissue 1. Homogenize tissue samples using a Cryogenic laboratory mill at −196 °C in liquid nitrogen according to Table 1.

Expected Results
The combination of methods for the proteomic screening and isolation of amyloid fibrils makes it possible to find and identify amyloid proteins in different organisms. In particular, we identified by the proteomic approach the list of proteins that form amyloidlike aggregates in the brain of Rattus norvegicus [8]. Using fibril immunoprecipitation, we proved that one of these proteins, RNA-binding protein FXR1, functions in the brain of rats and other jawed vertebrates in the amyloid form [8,10]. In this paper, we present fibrils of the FXR1 protein isolated from the brain of the red-eared turtle as an illustration ( Figure 3A). FXR1 fibrils isolated by immunoprecipitation are stained with Congo red and show yellow-green birefringence ( Figure 3B,C). We expect that similar results can be obtained using the described methodology for the detection of amyloids in any organisms with a sequenced and annotated genome. At least, using the PSIA-LC-MALDI method, amyloid-like proteins were identified in yeast, bacteria and pea seeds [6,7,9]. Moreover, the fibril immunoprecipitation method followed by Congo red staining provided direct evidence of the amyloid properties of yeast proteins Sup35 and Rnq1 in the strains containing [PSI + ] and [PIN + ] prions, respectively [19]. Slight differences in the protocols used for different organisms may be due to the peculiarities of tissue destruction and (or) the solubility of SDS-resistant protein aggregates. If necessary, for additional verification, fibrils isolated by immunoprecipitation can be stained with thioflavin S or T, as well as with conformation-dependent antibodies that recognize various amyloid fibrils and oligomers. Theoretically, the proteomic screening and immunoprecipitation of amyloid fibrils can be combined with quantitative mass spectrometry, but determination of the amount of protein in samples is not the task of this methodology.
In summary, the combination of proteomic screening for amyloids and the isolation of amyloid fibrils using immunoprecipitation opens broad perspectives for the search and identification of functional and pathological amyloids.

•
TBS. Add 800 mL of distilled water and a stir bar to a 1 L beaker, add 7.313 g NaCl. Stir until everything dissolves. Add 25 mL of Tris-HCl pH 7.6 and make volume up to 1 L with distilled water. TBS is stable at 4 °C for 3 months. We expect that similar results can be obtained using the described methodology for the detection of amyloids in any organisms with a sequenced and annotated genome. At least, using the PSIA-LC-MALDI method, amyloid-like proteins were identified in yeast, bacteria and pea seeds [6,7,9]. Moreover, the fibril immunoprecipitation method followed by Congo red staining provided direct evidence of the amyloid properties of yeast proteins Sup35 and Rnq1 in the strains containing [PSI + ] and [PIN + ] prions, respectively [19]. Slight differences in the protocols used for different organisms may be due to the peculiarities of tissue destruction and (or) the solubility of SDS-resistant protein aggregates. If necessary, for additional verification, fibrils isolated by immunoprecipitation can be stained with thioflavin S or T, as well as with conformation-dependent antibodies that recognize various amyloid fibrils and oligomers. Theoretically, the proteomic screening and immunoprecipitation of amyloid fibrils can be combined with quantitative mass spectrometry, but determination of the amount of protein in samples is not the task of this methodology.
In summary, the combination of proteomic screening for amyloids and the isolation of amyloid fibrils using immunoprecipitation opens broad perspectives for the search and identification of functional and pathological amyloids.